Epoxidation of carbon-carbon double bond with membrane bound peroxygenase

ABSTRACT

A method has been discovered for the epoxidation of a compound having at least one carbon-carbon double bond, the method involves reacting a compound having at least one carbon-carbon double bond, a solvent, an oxidant, and membrane bound peroxygenase. Also discovered is a method for preparing the membrane bound peroxygenase involving grinding seeds containing peroxygenase to produce ground seeds, homogenizing the ground seeds in a buffer to form a slurry, centrifuging the slurry to produce a first supernatant, centrifuging the first supernatant to produce a second supernatant, and filtering said second supernatant through a protein-binding membrane filter to produce membrane bound peroxygenase; optionally the second supernatant is filtered through a hydrophilic membrane filter prior to filtering the second supernatant through a protein-binding membrane filter.

BACKGROUND OF THE INVENTION

[0001] The present invention relates to a method for the epoxidation ofa compound having at least one carbon-carbon double bond, the methodinvolves reacting a compound having at least one carbon-carbon doublebond, a solvent, an oxidant, and membrane bound peroxygenase. Thepresent invention also relates to a method for preparing the membranebound peroxygenase involving grinding seeds containing peroxygenase toproduce ground seeds, homogenizing the ground seeds in a buffer to forma slurry, centrifuging the slurry to produce a first supernatant,centrifuging the first supernatant to produce a second supernatant, andfiltering said second supernatant through a protein-binding membranefilter to produce membrane bound peroxygenase; optionally the secondsupernatant is filtered through a hydrophilic membrane filter prior tofiltering the second supernatant through the protein-binding membranefilter.

[0002] Fats and oils (e.g., soybean oil and the esters of tall oil fattyacids) are renewable materials that are used as feed stocks for theproduction of industrial materials such as paints, varnishes,emulsifiers, and lubricants. To achieve a formulation with the desiredproperties, it is usually necessary to chemically modify the fat or oil.For unsaturated fats and oils, one common modification is epoxidationwhich leads to a material with increased polarity and enhancedreactivity (Carlson, K. D., et al., J. Am. Oil Chem. Soc., 62: 934-939(1985); Piazza, G. J., Some Recent Advances in Epoxide Synthesis, INRecent Developments in the Synthesis of Fatty Acid Derivatives, editedby G. Knothe and J. T. P. Derksen, AOCS Press, Champaign, 1999, pp.182-195)). Epoxidized oils are used as plasticizers and are generated onan industrial scale using peracids (Gan, L. H., et al., J. Am. Oil Chem.Soc., 69: 347-351 (1992)). The latter are generated by reacting formicor acetic acid with hydrogen peroxide in the presence of a strong acidcatalyst. A disadvantage of this procedure is that the strong acidcatalyzes epoxide ring opening, causes equipment corrosion, and it mustbe recycled or neutralized before discharge into the environment. Alsothe peracid intermediate is unstable, and explosive conditions arepossible.

[0003] The use of enzymes offers the possibility of developing anenvironmentally benign and more selective epoxidation reaction. Oneenzyme that might be useful for this purpose is termed peroxygenase.This enzyme catalyzes the heterolytic cleavage of a peroxygen bond andtransfers the liberated oxygen to an oxidizable functional group, suchas a carbon-carbon double bond, to give an epoxide product. Thus, in thepresence of organic hydroperoxide, oleic acid 1 is converted to the9,10-epoxide 2 by peroxygenase isolated from soybean, broad bean, andoat (FIG. 1) (Hamberg, M., et al., Arch. Biochem. Biophys., 283: 409-416(1990); Hamberg, M., et al., Plant Physiol., 110: 807-815 (1996);Hamberg, M., et al., Plant. Physiol., 99: 987-995 (1992); Blée, E., etal., J. Biol. Chem., 268:1708-1715 (1993); Blée, E., Phytooxylipins: ThePeroxygenase Pathway, IN Lipoxygenase and Lipoxygenase Pathway Enzymes,edited by G. J. Piazza, AOCS Press, Champaign, 1996, pp. 138-161)).Similarly linoleic acid afforded the 9,10-and 12,13-epoxy derivatives.Studies with peroxygenase from soybean and broad bean show thatcis-double bonds are the preferred substrates of peroxygenase (Hamberg,M., et al., Plant. Physiol., 99: 987-995 (1992); Blée, E., et al., J.Biol. Chem., 265: 12887-12894 (1990)). Peroxygenase can also catalyzeinternal epoxidation if the peroxygen group is contained in a moleculewith a double bond. Thus when the peroxygenases from soybean and broadbean were presented with the enzymatically-generated hydroperoxide oflinoleic acid 3 (13(S)-hydroperoxy-9(Z),11(E)-ocatdecadienoic acid,HPODE) products were 13(S)-hydroxy-9(Z),11(E)-octadecadienoic acid 4 and9,10-epoxy-13(S)-hydroxy-11(E)-octadecenoic acid 5 (Hamberg, M., et al.,Biochem. Biophys., 283: 409-416 (1990); Hamberg, M., et al., Arch.Biochem. Biophys., 283: 409-416 (1990); Blée, E., et al., J. Biol.Chem., 268:1708-1715 (1993); Piazza, G. J., et al., J. Am. Oil Chem.Soc., 76: 551-555 (1999)). Recently it has been demonstrated that aperoxygenase from oat seeds can catalyze the epoxidation of oleic acidusing hydrogen peroxide as the oxygen donor ( Hamberg, M., et al., PlantPhysiol., 110: 807-815 (1996)).

[0004] Traditional methods for using an enzyme for synthesis require twoseparate steps: isolation/purification of the enzyme from its biologicalsource and chemical or physical immobilization. In the study describedherein, a simple, rapid method for immobilizing oat seed peroxygenase onfilter membranes is described. The activity and reusability of theimmobilized peroxygenase preparation also was investigated, and the pHand temperature dependence of epoxidation by membrane-bound peroxygenasewas examined to determine optimal reaction conditions.

SUMMARY OF THE INVENTION

[0005] In accordance with the present invention there is provided amethod for the epoxidation of a compound having at least onecarbon-carbon double bond, the method involves reacting a compoundhaving at least one carbon-carbon double bond, a solvent, an oxidant,and membrane bound peroxygenase. There is also provided a method forpreparing the membrane bound peroxygenase involving grinding seedscontaining peroxygenase to produce ground seeds, homogenizing the groundseeds in a buffer to form a slurry, centrifuging the slurry to produce afirst supernatant, centrifuging the first supernatant to produce asecond supernatant, and filtering said second supernatant through aprotein-binding membrane filter to produce membrane bound peroxygenase;optionally the second supernatant is filtered through a hydrophilicmembrane filter prior to filtering the second supernatant through theprotein-binding membrane filter.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006]FIG. 1 shows two reactions catalyzed by peroxygenase; Top:formation of epoxide 2 with externally added hydroperoxide, Bottom:peroxygenase cleavage of fatty acid hydroperoxide 3 with and withoutinter-or intramolecular oxygen transfer to form alcohol 4 and epoxyalcohol 5;

[0007]FIG. 2 shows an assay in heptane on compound 3 in FIG. 1 ofperoxygenase residing on different membranes. Colorless bars, alcohol 4(methyl 13(S)-hydroxy-9(Z),11(E)-octadecadienoate); cross hatch bars,epoxy alcohol 5 (methylcis-9,10-epoxy-13(S)-hydroxy-11(E)-octadecenoate). Values are the mean±SE,n=3-6. Nylon-1: Nylon 66, 0.45 μm; Nylon-2: Nylon 66, 0.2 μm;Durapore-1: Durapore, 0.22 μm; Durapore-2: Durapore,0.1 μm; Durapore-3:Durapore hydrophobic,0.22 μm. Other membranes were as described below.Each assay also contained Me-HPODE (10 mg) and 0.7 mL water-saturatedheptane.

[0008]FIG. 3 shows the time course of epoxidation of oleic and elaidicacids by peroxygenase bound on a Fluoropore membrane. Assays contained 5mg (17.7 μmol) oleic acid () or elaidic acid (▪) and 7.0 mLbuffer/Tween. At t=0, 1, 2, and 4 h, 7.3 μmol t-butyl hydroperoxide wasadded. At t=6 h, 21.9 μmol t-butyl hydroperoxide was added.

[0009]FIG. 4 shows the effect of pH on the epoxidation of sodium oleateby peroxygenase bound on a Fluoropore membrane. Assays contained 5 mg(16.4 μmol), 6.3 ml buffer, 0.7 ml 1% (w/v) Tween 20, and 7.3 μmolt-butyl hydroperoxide () or H₂O₂ (▪). Assays were conducted for 1 h at20° C. Percent yield calculations based on sodium oleate (theoreticalmaximum 44%). Each data point is the average of four to six repetitions.

[0010]FIG. 5 shows the effect of temperature on the epoxidation ofsodium oleate by peroxygenase bound on a Fluoropore membrane. Assayswere conducted at pH 7.5 for t-butyl hydroperoxide () and pH 5.5 forH₂O₂ (▪). Other assay conditions as in FIG. 4.

DETAILED DESCRIPTION OF THE INVENTION

[0011] A method is disclosed for the epoxidation of a compound having atleast one carbon-carbon double bond. It is expected that the method maybe used for the epoxidation of any compound having at least onecarbon-carbon double bond; examples of such compounds (e.g., olefiniccompounds) are found in the following U.S. Pat. Nos. (which areincorporated by reference in their entirety): 6,160,138; 5,780,655;5,620,938; 5,354,875. The compound having a carbon-carbon double bondgenerally includes alkenes (e.g., C_(n)H_(2n) where n is two to about100, preferably n is two to about 20, more preferably n is about six toabout 18; preferably the cis stereoisomers of the alkenes), the abovealkenes that are substituted (e.g., R—CH═C—R′ where R and R′ are anaromatic or aliphatic organic compound such as —C₆H₅,—C₂H₅,—C₃H₇; R andR′ may also contain a heteroatom substituent such as —C₃H₆X where X isOH, Cl, F, Br, NH₂,SH, or COOH, or —C₃H₅X where X is O or NH; R and R′may be the same or different), compounds having more than onecarbon-carbon double bond (e.g., R—CH₂(CH═CH)_(x)(CH₂)_(y)CH═CHCH₂R′where x is one to about 100 (preferably one to about 20, more preferablyone to about five), where y is zero to about 100 (preferably zero toabout 14, more preferably zero to about three), R and R′ are as definedabove or may also be H), unsaturated fatty acids (e.g., oleic acid,linoleic acid, myristoleic acid, palmitoleic acid, vaccenic acid,ricinoleic acid, conjugated linoleic acid, linolenic acid, gammalinolenic acid, eicosenoic acid, eicosadienoic acid, eicosatrienoicacid, arachidonic acid, erucic acid, brassidic acid, docosahexaenoicacid), and derivatives of unsaturated fatty acids (e.g., esterderivatives such as methyl oleate, ethyl oleate, isopropyl oleate,lauryl oleate, myristyl oleate, palmityl oleate, stearyl oleate, methyllinoleate, ethyl linoleate, isopropyl linoleate, lauryl linoleiate,myristyl linoleate, palmityl linoleate, stearyl linoleate, methylated13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate; amide derivatives such asoleamide, linoleamide, methyl oleamide, methyl linoleamide, ethyloleamide, ethyl linoleamide, lauryl oleamide, lauryl linoleamide,myristyl oleamide, myristyl linoleamide, palmityl oleamide, palmityllinoleamide, oleayl oleamide, oleayl linoleamide). The present inventionmay be used to produce ethylene oxide, propylene oxide, andepichlorohydrin. Preferred compounds having at least one carbon-carbondouble bond include those with cis double bonds (e.g., those describedin U.S. Pat. No. 6,160,138 where either R₁ and R₃ or R₂ and R₄ arehydrogen), terminal double bonds (e.g., styrene), and those with acyclic structure (e.g., those described in U.S. Pat. No. 6,160,138).

[0012] The method involves reacting a compound having at least onecarbon-carbon double bond and membrane bound peroxygenase in thepresence of a solvent and an oxidant. The solvent may be an aqueoussolvent composed of a buffer having a pH from about 4.5 to about 9.5(preferably a pH from about 5 to about 9) such as aqueous phosphatebuffer/Tween or water with a manual or automatic monitoring system thatdetects and adjusts the pH or hydrogen ion concentration; the solventmay be a nonpolar solvent such as heptane, isooctane, dichloromethane,or toluene. The reaction time is about one minute up to about seven days(preferably about two hours to about 24 hours, more preferably about sixhours to about 12 hours). The reaction temperature is about 5° C. toabout 75° C. (preferably about 15° C. to about 65° C., more preferablyabout 15° C. to about 35° C.). The oxidant is generally an organichydroperoxide such as t-butyl hydroperoxide or hydrogen peroxide;examples of oxidants include t-butyl hydroperoxide (max yield at pH7.5-8.0), cumene hydroperoxide, hydrogen peroxide (max yield at pH 5.5),and urea-hydrogen peroxide complex. Generally, the oxidant is added insteps instead of all at once since it was found that the addition oflarge amounts of some oxidizing agents tends to diminish the activity ofperoxygenase. A convenient method is to add (generally manually) 20 mol% of the oxidant every one or two hours; an automatic addition of smallamounts of oxidant more frequently is possible. If the oxidant is foundto be toxic to the peroxygenase enzyme activity, then the oxidant may beadded even more slowly to insure that its concentration in the reactionmedium remains low. As the reaction progresses, the rate of conversionof starting material to product slows, and the oxidant may be added lessfrequently, or alternatively if automatic addition is used then theoxidant may be added more slowly. The total amount of oxidant added toachieve maximum conversion to epoxide is 140 to 290 mole % of startingmaterial, although for some applications incomplete conversion toepoxide is desirable (in which case less oxidant is added).

[0013] One unexpected advantage of the present invention is the ease inisolating the epoxidized compound. At the end of the epoxidationreaction, the reaction medium is physically separated from themembrane-bound peroxygenase: This is achieved in the laboratory bydecanting the reaction medium from the membranes, and then washing themembranes with diethyl ether to remove traces of epoxide from themembranes. If the reaction medium is an organic solvent such as heptane,the organic solvent and diethyl ether wash are combined and then removedby evaporation to give the epoxide product. If the reaction medium isaqueous and a fatty acid is used as the starting material, the reactionmedium is treated with dilute hydrochloric acid to give pH 3.0. Theacidified medium is extracted with diethyl ether. The diethyl etherextract and the diethyl ether fraction from the membrane wash arecombined and evaporated to give the epoxide product. If the reactionmedium is aqueous and the ester of a fatty acid is used as the startingmaterial or the starting material consists of a substance that containsno carboxylic acid, such as cyclohexene or styrene, then theacidification step is omitted.

[0014] The membrane bound peroxygenase is produced by a method involvinggrinding seeds (e.g., oat, soybean, broad bean) containing peroxygenase,generally for about 20 to about 60 seconds, to produce ground seeds;homogenizing the ground seeds in a buffer (generally a buffer having apH near 7 (e.g., about 6.5 to about 7.5), specifically 0.1 M potassiumphosphate having a pH of 6.7) to form a slurry, generally homogenizinginvolves blending for about one to about two minutes; centrifuging theslurry to produce a first supernatant, generally at about 9,000 to about16,000×g for about 10 to about 15 min.; centrifuging the firstsupernatant to produce a second supernatant, generally at about 9,000 toabout 16,000×g for about 10 to about 15 min.; and filtering the secondsupernatant through a protein-binding membrane filter to producemembrane bound peroxygenase. The protein-binding membrane filter musthave a relatively small pore size (about 0.1 to about 2 μm, preferablyabout 0.2 to about 1 μm ) in order to effectively hold the peroxygenaseenzyme; this is extremely important since it was not predictable thatthe enzyme would be held onto this type of membrane, that it would beactive on this membrane, and that it would not “wash” off of thismembrane when the membrane was added to an aqueous reaction mixture.Generally the protein-binding membrane filter includes Nylon 66 (0.2 μm)and hydrophobic membranes such as Fluoropore (polytetrafluoroethylene(PTFE); 0.2 μm) and Durapore (0.22 μm) membrane filters. Optionally thesecond supernatant is prefiltered through a hydrophilic membrane filterprior to filtering the second supernatant through the protein-bindingmembrane filter. Generally the hydrophilic membrane filter will filterout the extraneous matter without holding onto the desired peroxygenaseactivity; the necessary pore size of such hydrophilic membrane filtersis easily determined by one skilled in the art. Such hydrophilic(water-loving or polar) membranes include Durapore (polyvinylidienefluoride (PVDF)).

[0015] The following examples are intended only to further illustratethe invention and are not intended to limit the scope of the inventionas defined by the claims.

EXAMPLES

[0016] Oat seeds (Avena sativa L.) were supplied by Equine SpecialityFeed Co. (Ada, Minn.). Durapore (PVDF, hydrophilic) membranes andFluoropore (PTFE, hydrophobic) membranes were from Millipore (Bedford,Mass.). Sodium oleate was purchased from Nu-Chek-Prep, Inc. (Elysian,Minn). Heptane and hydrogen peroxide (30%) were purchased from Aldrich(Milwaukee, Wis.). Sigma (St. Louis, Mo.) was the source of t-butylhydroperoxide (70%). Water was purified to a resistance of 18 mΩ-cmusing a Barnstead (Dubuque, Iowa) NANO pure system. All other reagentswere of the highest purity available. Preparation of 3 methyl13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate (Me-HPODE): Linoleic acidwas enzymatically converted to HPODE using lipoxygenase as describedpreviously (Piazza, G. J., et al., J. Am. Oil Chem. Soc., 74: 1385-1390(1997)) and HPODE was methylated with CH₂N₂ to give Me-HPODE.

[0017] Preparation of oat seed microsomes (membrane-bound peroxygenase):For small scale reactions, dry oat seeds (10 g) were ground in 5 gbatches in a 37 mL Waring Blender (New Hartfod, Conn.) mini-jar for 30s. The ground oat seeds were transferred to a 110 mL mini-jar containing90 mL cold 0.1 M potassium phosphate buffer (pH 6.7) and blended for 90s at high speed. The oat seed slurry was centrifuged at 9000×g for 10min. The pellet was discarded and the supernatant was centrifuged for anadditional 10 min at 9000×g. After the second centrifugation, the pelletwas discarded and the supernatant was divided into four equal portionsand each portion was subjected to vacuum infiltration with a Fluoroporemembrane (0.2 μm, 47 mm). The Fluoropore membrane was wetted withmethanol before loading onto the membrane holder. After vacuuminfiltration, the membrane was cut into four equal size pieces, andthese pieces placed into a reaction flask.

[0018] For large scale reactions 100 g of oat were ground dry (30 s),and then the ground seeds were homogenized in 900 mL of cold 0.1 Mpotassium phosphate buffer (pH 6.7) using a Waring commercial blender (2min). The slurry was centrifuged at 16,000×g for 15 min. The pellet wasdiscarded and the supernatant was centrifuged for an additional 15 minat 16,000×g. The supernatant was passed through a hydrophilic Duraporemembrane filter (0.65 μm, 142 mm), and the filtrate was collected anddivided into quarters and each was passed through a hydrophobicFluoropore membrane (0.2 μm, 142 mm).

[0019] Epoxidation reactions: The indicated amount of Me-HPODE, oleicacid, or elaidic acid dissolved in CH₂Cl₂ was added to a 10 or 15 mLstoppered Erlenmeyer flask, and the solvent removed under a stream ofnitrogen. Into each flask was added water-saturated heptane or 0.1 Mpotassium phosphate buffer (pH 6.7) containing 0.1% (w/v) Tween 20 andthe membrane pieces. The reaction was initiated by adding t-butylhydroperoxide. The suspension was agitated at 20° C. for 2 h or asindicated. At the end of the incubation period, 3.5 mL methanol wasadded, and after removal of the membrane pieces, the contents weretransferred to a 125 mL separatory funnel. The products were partitionedbetween 30 mL diethyl ether and 25 mL water. After separating thelayers, the water layer was extracted with 25 mL diethyl ether. Theether fractions were combined, dried over sodium sulfate, and taken todryness under a stream of nitrogen. The products were dissolved in 2 mLdichloromethane and stored at −20° C. until analysis. When fatty acidwas the substrate, the products were methylated with CH₂N₂ beforeanalysis.

[0020] Epoxidations: Smaller scale reactions, used for determining pHand temperature profiles, contained 5 mg (16.4 μmol) sodium oleate, 6.3mL buffer, 0.7 mL 1% (w/v) Tween 20, and 7.3 μmol t-butyl hydroperoxideor H₂O₂. The buffer consisted of four components, each with a differentpKa, to provide buffering capacity over a broad pH range. Each componentwas present at a concentration of 50 mM. The components were tricine(N-tris(hydroxymethyl)-methylglycine), MES(2-(N-morpholino)ethanesulfonic acid), HEPES(N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)), and glacialacetic acid. Each assay was agitated at 20° C. for 1 h or as indicated.Larger scale reactions contained 100 mg (0.329 mmol) sodium oleate, and32.4 mL buffer. The buffer consisted of 50 mM HEPES/0.1% (w/v) Tween 20,pH 7.5, for reactions using t-butyl hydroperoxide or 50 mM MES/ 50 mMglacial acetic acid/0.1% (w/v) Tween 20, pH 5.5, for reactions usinghydrogen peroxide. Products were extracted and analyzed by HPLC (Piazza,G. J., et al., Biotech. Lett., 22: 217-221 (2000)) as described below.

[0021] High performance liquid chromatography (HPLC): Reaction productswere separated on a Lichrosorb 5μ diol HPLC column (250×10 mm)(Phenomenex, Torrance, Calif.) installed on a Waters (Milford, Mass.)LCM1 HPLC instrument. The instrument was equipped with a Waters 996photodiode array detector in tandem with a Varex evaporativelight-scattering detector MK III (Alitech, Deerfield, Ill.) operated ata temperature of 55° C., and with N₂ as the nebulizing gas at a flowrate of 1.5 L/min. Mobile phase composition and gradient was hexane:isopropanol (97:3) to (94:6) over 29 min using a linear gradient. Theflow rate was 2 mL/min.

[0022] Gas chromatography-mass spectrometry (GC-MS): Mass spectra wereobtained on a Hewlett Packard (HP) (Wilmington, Del.) 5890 Series IIPlus gas chromatograph equipped with a HP 5972 mass selective detectorset to scan from m/e 45 to 400 at 2 scans per s. A capillary column(Supelco SP-2340, 60 m×0.25 mm) coated with 0.20 μmpoly(biscyanopropylsiloxane) was used to separate the products. The oventemperature was increased from 130° C. to 177° C. at 2° C. per min andheld at 177° C. for 5 min; increased to 230° C. at 10° C. per min andheld at 230° C. for 15 min; increased to 250° C. at 10° C. per min andheld at 250° C. for 10 min. The injector port temperature was 250° C.,and the detector transfer line temperature was 250° C.

[0023] Oat seeds were homogenized in buffer, and after two low speedcentrifugations, the resulting supernatant was passed through filterpapers or membranes. The amount of peroxygenase activity retained on thefilter papers or membranes was determined by measuring the conversion ofhydroperoxide 3 to products 4 and 5 by HPLC (FIG. 1). No activity wasobserved with Whatman paper filters tested whereas all tested membranesshowed some retained peroxygenase activity (FIG. 2). With each membrane,more alcohol 4 formed than epoxy alcohol 5. The highest levels of 5 wereobtained with the Durapore and Fluoropore membranes which are bothhydrophobic. Further experimentation was performed only with theFluoropore membrane.

[0024] As shown in FIG. 2, an unsaturated substrate can be converted toan epoxide by peroxygenase when an appropriate oxidant is present. Table1 lists the results obtained with four different oxidants used on oleicacid. When aqueous phosphate buffer/Tween was the solvent, t-butylhydroperoxide and cumene hydroperoxide gave better yields of epoxidethan hydrogen peroxide or its urea adduct. A similar finding wasobtained in the solvent heptane except that the overall yields ofepoxide were somewhat lower. Contol experiments with the oxidants, oleicacid, and Fluoropore membranes showed that no epoxide was formed withoutthe addition of the oat seed fraction. TABLE 1 Influence of solvent andoxidant on the epoxidation of oleic acid by peroxygenase bound on aFluoropore membrane. Epoxide (μmol)^(a) (% yield of epoxide) OxidantAqueous^(b) Heptane^(c) t-Butyl Hydroperoxide 2.8 (79%) 1.8 (51%) CumeneHydroperoxide 2.0 (56%) 1.6 (45%) Hydrogen Peroxide 0.6 (17%) 0.6 (17%)Urea-Hydrogen Peroxide 0.7 (20%) 0.5 (14%)

[0025] To ascertain whether membrane-bound peroxygenase is reusable, itis necessary to conduct repeat batch epoxidations. Table 2 shows theconversion of oleic acid to epoxide upon reuse of peroxygenaseimmobilized on a Fluoropore membrane. In the first cycle higher amountsof epoxide were obtained in buffer/Tween than in heptane, as before. Inthe second and third cycles lower amounts of epoxide were obtained, butin buffer/Tween and heptane the membrane preparation retained aconsiderable amount of its initial activity. By the third use, theamount of epoxide obtained was reduced by only 17% in buffer/Tween and38% in heptane. The very fact that Fluoropore-bound peroxygenaseretained its activity through three cycles in buffer/Tween demonstratesthat peroxygenase is not easily “washed” off. This was a surprisingresult given that peroxygenase was initially solubilized in aqueousbuffer. Without being bound by theory, the fact that peroxygenase wasnot washed off must indicate a strong degree of interaction betweenperoxygenase and the membrane. TABLE 2 Reuse of peroxygenase bound onFluoropore membrane for epoxidation of oleic acid with t-butylhydroperoxide. Epoxide (μmol)^(a) (% yield of epoxide) Cycle Aqueous^(b)Heptane^(c) 1 2.9 (82%) 1.6 (45%) 2 2.7 (76%) 1.4 (40%) 3 2.4 (68%) 1.0(28%)

[0026] When the amounts of oleic acid substrate and t-butylhydroperoxide oxidant were increased leaving the level of peroxygenaseimmobilized on a Fluoropore membrane unchanged, the percent yield of theepoxide of oleic acid decreased. For example from Tables 1 and 2, thepercent yield of epoxide obtained in aqueous buffer is approximately80%. When the level of oleic acid and t-butyl hydroperoxide wereincreased five-fold, the percent yield of epoxide was reduced toapproximately 60% even though the reaction time was increased from two hto twenty-four h. It was assumed that peroxygenase was deactivated byt-butyl hydroperoxide, as deactivation by H₂O₂ and HPODE has previouslybeen reported (Hamberg & Hamberg 1990b; 1992), Arch. Biochem. Biophys.283: 409-416 (1990) and Hanburg et al. Plant Physiol. 99: 987-995(1992). It was hypothesized that if smaller amounts of t-butylhydroperoxide were added several times over a longer time period,peroxygenase deactivation might be reduced. This was found to be thecase. As shown in FIG. 3, when smaller amounts of t-butyl hydroperoxidewere added over a 4 h period, 85% of oleic acid was converted to itsepoxide at 24 h.

[0027] When experiments were performed with elaidic acid, the transanalogue of oleic acid, the yield of epoxide was relatively low. At 24 honly 11% of elaidic acid was converted to its epoxide (FIG. 3).Discrimination against trans double bonds has been previously reportedfor peroxygenase isolated from soybean and broad bean (Hamberg, M., etal., Plant Physiol., 99: 987-995 (1992); Blée, E., et al., J. Biol.Chem., 265: 12887-12894 (1990)).

[0028] Thus a method for the isolation of peroxygenase has been devised.The peroxygenase preparation is reusable to a degree and can be used toconvert double bonds to epoxides. Although the example above emphasizedthe use of lipid substrates, it is expected that peroxygenase can be auseful catalyst for epoxide formation in a wide variety of chemicalsubstances. It is particularly useful in those situations requiring theabsence of an acidic catalyst or those in which the selectiveepoxidation of a cis double bond is required.

[0029] Optimizing reaction parameters: Before proceeding to larger scalereactions, it was necessary to determine reaction conditions thatpromoted the most rapid rate of epoxide formation. To accomplish this,reaction time was restricted to 1 h and suboptimal (less thanstoichiometric) quantities of oxidant added, as prior work indicatedthat higher oxidant levels deactivate peroxygenase (11-13). FIG. 4 showsthe affect of buffer pH on the yield of epoxide formed from sodiumoleate in one h. The highest yield of epoxide was obtained with t-butylhydroperoxide at pH 7.5-8.0. Hydrogen peroxide gave the highest yield ofepoxide at pH 5.5. Note however that the yield of epoxide wasapproximately identical for both t-butyl hydroperoxide and hydrogenperoxide at their pH optima.

[0030]FIG. 5 shows the influence of temperature on the yield in one hourreactions. When the oxidant t-butyl hydroperoxide was used, the yield ofepoxide increased as the temperature was increased to 65° C. and thendecreased slightly at 75° C. The yield of epoxide at higher temperatureswas the maximum possible given the amount of t-butyl hydroperoxideadded. In contrast, increasing the temperature when hydrogen peroxidewas the oxidant resulted in only modest increases in the epoxide yield,and at 75° C., the epoxide yield decreased. Note however that at 25° C.,the yield of epoxide was approximately the same for both t-butylhydroperoxide and hydrogen peroxide.

[0031] Larger scale reactions: Oat seeds and sodium oleate wereincreased twenty-fold. Readily available commercial filter holders couldaccommodate 142 mm (diameter) membranes. These are approximatelynine-fold higher in area than the 42 mm membranes used for the smallerscale reactions. It was found that membrane clogging was a problem, andaccordingly ways to reduce the particulate matter in the preparationswas sought. The centrifugation force was gradually increased. After eachincrease, a portion was passed through a Fluoropore membrane, and thiswas tested for epoxidation activity. It was found that thecentrifugation force could be increased from 9000×g to 16,000×g(r_(average)) without large losses in epoxidation activity. In addition,a prefiltration step with a 0.65 μm hydrophilic Durapore membrane wasadded, as our prior work has shown only minimal binding of peroxygenaseto this membrane (Piazza, G. J., et al., Biotech. Lett., 22: 217-221(2000)). As noted before, the addition of large amounts of oxidizingagent tends to diminish the activity of peroxygenase, and thereforeduring the preparation of epoxide the oxidizing agent was added insteps. Shown in Table 3 are the results of selected experiments with 100mg sodium oleate and t-butyl hydroperoxide chosen to illustrate thetrends that were observed. TABLE 3 Larger Scale Production of Epoxidewith t-Butyl Hydroperoxide and Hydrogen Peroxide Time (h) Reaction AReaction B Reaction C Reaction D t-Butyl Hydroperoxide^(a) or H₂O₂ ^(b)Added (μmol) 270 135 67.5 33.8 1 270 135 67.5 33.8 2 270 135 67.5 33.8 4270 135 67.5 33.8 6 810 405 203 101 Total 1890 946 473 236 Epoxide Yieldat 24 h^(c) (μmol) t-BuOOH 191 258 262 230 H₂O₂ 33.9 42.0 74.9 108Percent Yield Based on Sodium Oleate t-BuOOH 58.2 78.4 79.5 69.8 H₂O₂10.0 12.8 22.8 33.0 Percent Yield Based on t-Butyl Hydroperoxide or H₂O₂t-BuOOH 10.1 27.3 55.4 97.4 H₂O₂ 1.7 4.4 15.8 45.8

[0032] In Table 3, at the lowest level of added oxidatant (Reaction D),the utilization of hydroperoxide was nearly quantitative, giving a 97.4%yield based upon hydroperoxide. However, when the amount of addedt-butyl hydroperoxide was elevated (Reactions B and C) to achieve betterconversion of sodium oleate, the yield of epoxide based upon sodiumoleate could be increased only to about 80%, a yield similar to thatachieved with 1 mg sodium oleate in our prior research with peroxygenase(Piazza, G. J., et al., Biotech. Lett., 22: 217-221 (2000)). Asillustrated with Reaction A, further increases in t-butyl hydroperoxidediminished the yield of epoxide. When hydrogen peroxide was used as theoxidant, the highest yield of epoxide, 33% based on sodium oleate, wasobtained at the lowest level of added hydrogen peroxide (Reaction D). Asthe level of added hydrogen peroxide was increased, the yield of epoxidegradually decreased. Thus hydrogen peroxide is highly toxic toperoxygenase.

[0033] In conclusion, the successful scale-up of peroxygenase catalyzedepoxidation has been achieved using t-butyl hydroperoxide as theoxidant. We expect that further increases in scale are likely usingt-butyl hydroperoxide and other oxidants.

[0034] Epoxidation of styrene and cyclohexene: Reaction mixturescontained 1.9 mg (18.2 μmol) styrene or 1.5 mg (18.2 μmol) cyclohexene,7.0 mL 50 mM Hepes/0.1% (w/v) Tween 20, pH 7.5, and peroxygenaseimmobilized on a 47 mm, 0.2 μm Fluoropore membrane. At 0, 1, 2, and 4 h,3.34 μmol t-butyl hydroperoxide was added; at 6 h, 20.0 μmol t-butylhydroperoxide was added. The reaction temperature was 25° C. At 24 h asample was removed using a solid phase microextractor with apolydimethyl siloxane filter (100 μm thickness)( Supelco, Bellefonte,Pa.) and analyzed on an HP-MS 5890 GC/MS containing a 30 m HP-5MS column(Hewlett Packard, Palo Alto, Calif.). Epoxide yields shown below wereobtained from separately prepared reaction: Styrene Cyclohexene (PercentYield of Epoxide) 60.0 76.5 60.8 72.8

[0035] The above example shows that the peroxygenase can be a usefulcatalyst for epoxide formation in a wide variety of chemical substancesbesides lipid substrates.

[0036] All of the references and U.S. patents cited herein areincorporated by reference in their entirety.

[0037] Thus, in view of the above, the present invention concerns (inpart) the following:

[0038] A method for preparing membrane bound peroxygenase, said methodinvolving (comprising, consisting essentially of, consisting of)grinding seeds containing peroxygenase to produce ground seeds,homogenizing the ground seeds in a buffer to form a slurry, centrifugingthe slurry to produce a first supernatant, centrifuging the firstsupernatant to produce a second supernatant, and filtering the secondsupernatant through a protein-binding membrane filter to producemembrane bound peroxygenase.

[0039] The above method, further involving optionally filtering thesecond supernatant through a hydrophilic membrane filter prior tofiltering the second supernatant through the protein-binding membranefilter.

[0040] The above method, wherein the seeds are oat, soybean, broad beanor mixtures thereof.

[0041] The above method, wherein the grinding involves grinding forabout 20 to about 60 seconds.

[0042] The above method, wherein the homogenizing involves blending forabout one to about two minutes.

[0043] The above method, wherein the buffer has a pH of about 7 (e.g.,0.1 M potassium phosphate buffer at pH 6.7).

[0044] The above method, wherein the centrifuging of the slurry and thefirst supernatant is at about 9,000 to about 16,000×g for about 10minutes to about 15 minutes.

[0045] The above method, wherein the protein-binding membrane has a poresize of about 0.1 μm to about 2 μm.

[0046] Membrane bound peroxygenase prepared by the above method.

[0047] A method for the epoxidation of a compound having at least onecarbon-carbon double bond, the method involving (comprising, consistingessentially of, consisting of) reacting a compound having at least onecarbon-carbon double bond, a solvent, an oxidant, and membrane boundperoxygenase prepared by the above method.

[0048] The above method, wherein the solvent is an aqueous solventhaving a pH from about 4.5 to about 9.5 or a nonpolar solvent.

[0049] The above method, wherein the oxidant is an organic hydroperoxide(e.g., t-butyl hydroperoxide or hydrogen peroxide).

[0050] The above method, wherein the reaction time is about one minuteup to about seven days.

[0051] The above method, wherein the reaction time is about two hours toabout 24 hours.

[0052] The above method, wherein the reaction time is about six hours toabout 12 hours.

[0053] The above method, wherein the reaction temperature is about 5° C.to about 75° C.

[0054] The above method, wherein the reaction temperature is about 15°C. to about 65° C.

[0055] The above method, wherein the reaction temperature is about 15°C. to about 35° C.

[0056] The above method, further comprising isolating (recovering) theepoxidized compound.

[0057] Other embodiments of the invention will be apparent to thoseskilled in the art from a consideration of this specification orpractice of the invention disclosed herein. It is intended that thespecification and examples be considered as exemplary only, with thetrue scope and spirit of the invention being indicated by the followingclaims.

We claim:
 1. A method for preparing membrane bound peroxygenase, saidmethod comprising grinding seeds containing peroxygenase to produceground seeds, homogenizing said ground seeds in a buffer to form aslurry, centrifuging said slurry to produce a first supernatant,centrifuging said first supernatant to produce a second supernatant, andfiltering said second supernatant through a protein-binding membranefilter to produce membrane bound peroxygenase.
 2. The method accordingto claim 1, further comprising optionally filtering said secondsupernatant through a hydrophilic membrane filter prior to filteringsaid second supernatant through said protein-binding membrane filter. 3.The method according to claim 1, wherein said seeds are selected fromthe group consisting of oat, soybean, broad bean and mixtures thereof.4. The method according to claim 1, wherein said grinding comprisesgrinding for about 20 to about 60 seconds.
 5. The method according toclaim 1, wherein said homogenizing comprises blending for about one toabout two minutes.
 6. The method according to claim 1, wherein saidbuffer has a pH of about
 7. 7. The method according to claim 1, whereinsaid centrifuging of said slurry and said first supernatant is at about9,000 to about 16,000×g for about 10 minutes to about 15 minutes.
 8. Themethod according to claim 1, wherein said protein-binding membrane has apore size of about 0.1 μm to about 2 μm.
 9. Membrane bound peroxygenaseprepared by the method according to claim
 1. 10. A method for theepoxidation of a compound having at least one carbon-carbon double bond,said method comprising reacting a compound having at least onecarbon-carbon double bond, a solvent, an oxidant, and membrane boundperoxygenase prepared by the method according to claim
 1. 11. The methodaccording to claim 10, wherein said solvent is an aqueous solvent havinga pH from about 4.5 to about 9.5 or a nonpolar solvent.
 12. The methodaccording to claim 10, wherein said oxidant is an organic hydroperoxide.13. The method according to claim 10, wherein the reaction time is aboutone minute up to about seven days.
 14. The method according to claim 10,wherein the reaction time is about two hours to about 24 hours.
 15. Themethod according to claim 10, wherein the reaction time is about sixhours to about 12 hours.
 16. The method according to claim 10, whereinthe reaction temperature is about 5° C. to about 75° C.
 17. The methodaccording to claim 10, wherein the reaction temperature is about 15° C.to about 65° C.
 18. The method according to claim 10, wherein thereaction temperature is about 15° C. to about 35° C.
 19. The methodaccording to claim 10, further comprising isolating the epoxidizedcompound.